The secondary structure of DNA is variable and depends on the sequence of nu- cleotides in a strand. While DNA can form duplexes, formations of three, four, or even a single strand have been observed in vivo and in vitro as well. In this thesis, we study the effect of small changes of oligonucleotide sequences on the stability of hairpins formed by DNA heptamers by 1 H nuclear magnetic resonance (NMR) spectroscopy. Suitable DNA sequences were selected based on symmetry rules and stability prediction by near- est neighbor model. Two-dimensional 1 H -1 H NOESY spectra were used to assign the 1 H resonances of aromatic hydrogens. Variable-temperature 1D spectra served for ob- taining melting curves, from which the thermodynamic properties of the hairpins were determined. The presence of hairpins in the solutions was confirmed by the character of the NOESY spectra, independence of melting temperature on oligonucleotide concen- tration, and comparison of competing melting-curve models of duplex and hairpin. Our results point out the importance of the order of the stem base pairs and contribute to the description of the extraordinary stability of DNA mini-hairpins. 1

<p>DNA interstrand crosslinks provide a challenge for repair machinery given that both strands contain the lesion. Cells have evolved a sophisticated mechanism to overcome this, by recruiting proteins from several repair pathways. One protein thought to function solely in interstrand-crosslinking repair is Pso2. Pso2 deficient cells display sensitivity towards ICL agents and accumulate DNA double strand breaks upon exposure. However, Pso2 is not required for repair of DNA double strand breaks generated by other means, suggesting that these particular breaks are unique requiring Pso2 processing for successful repair. To identify what characteristics these breaks possess and what role Pso2 plays in processing theses breaks, a thorough <em>in vivo</em> and <em>in vitro </em>characterization of Pso2 was conducted.</p> <p>Pso2 was found to be a 5’-exonuclease independent of DNA structure and length but completely dependent on a 5’-phosphate. Pso2 also displayed structure-specific DNA hairpin-opening activity at the 3’ end two nucleotides from the apex. This activity was required for repair of genomic DNA capped by hairpin structures in the absence of ICL inducing agents as well those generated in response to ICL damage. The constitutively active DNA hairpin endonuclease β-CASP domain of Artemis was able to partially restore the DNA hairpin-opening deficiency and suppress the ICL defect in a <em>pso2 </em>null strain. This suggests that Pso2 acts as an endonuclease in ICL repair and that DNA hairpins may be an encountered intermediate, leading to further understanding of how this unique protein function in ICL repair as well as the repair mechanism itself.</p> / Doctor of Science (PhD)

<p>Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, <i>attI </i>and<i> attC</i> resulting in excision and integration of short DNA elements called gene cassettes carrying an <i>attC</i> site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The<i> attC</i> of the <i>aadA1-qacE</i> cassette junction in Tn<i>21</i> was characterized in detail. Like other <i>attC</i> sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of <i>attC</i>. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among <i>attC</i> sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of <i>attC</i> hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.</p>

Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, attI and attC resulting in excision and integration of short DNA elements called gene cassettes carrying an attC site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The attC of the aadA1-qacE cassette junction in Tn21 was characterized in detail. Like other attC sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of attC. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among attC sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of attC hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.

The synthesis of four homologous anthraquinones (AQ I-IV) bearing increasing lengths of polyethylene glycol (PEG) side chains and their binding to AT- and GC-rich DNA hairpins are reported. The molecules were designed such that the cationic charge is at a constant position and the ethylene glycol units chosen to allow significant increases in size with minimal changes in hydrophobicity. The mode and affinity of binding were assessed using circular dichroism (CD), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). The binding affinity decreased as the AQ chain length increased along the series with both AT- and GC-rich DNA. ITC measurements showed that the thermodynamic parameters of AQ I-IV binding to DNA exhibited significant enthalpy-entropy compensation. The enthalpy became more favorable while the entropy became less favorable. The correlation between enthalpy and entropy may involve not only the side chains, but also changes in the binding of water and associated counterions and hydrogen bonding. The interactions of AQ I-IV with GC-rich DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The kinetics of a homologous series of anthraquinone threading intercalators, AQT I-IV with calf thymus DNA was studied using the stopped-flow. The threading mechanisms of the anthraquinones binding to DNA showed sensitivity to their side chain length. Fitting of the kinetic data led to our proposal of a two step mechanism for binding of AQT I, bearing the shortest side chain, and a three step mechanism for binding of the three longer homologs. Binding involves formation of an externally bound anthraquinone-DNA complex, followed by intercalation of the anthraquinone for AQT I-IV, then isomerization to another complex with similar thermodynamic stability for AQT II-IV.

No description available.

Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurements. / Jeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird.

Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurements. / Jeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird.